Spatial proteomics in three-dimensional intact specimens

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of biological specimens imaged in 3D as a whole are lacking. Here, we present DISCO-MS, a technology combining whole-organ/ism imaging, deep learning-based image analysis, robotic tissue extraction and ultra-high sensitivity mass spectrometry. DISCO-MS yielded qualitative and quantitative proteomics data indistinguishable from uncleared samples in both rodent and human tissues. Using DISCO-MS, we investigated microglia activation along axonal tracts after brain injury and characterized early and late-stage individual amyloid-beta plaques in Alzheimer's disease mouse model. Furthermore, aided by DISCO-bot robotic extraction we studied regional proteomics heterogeneity of immune cells in intact mouse bodies and aortic plaques in whole human heart. Overall, DISCO-MS enables unbiased proteome analysis of pre-clinical and clinical tissues after unbiased imaging of entire specimens in 3D, providing new diagnostic and therapeutic opportunities for complex diseases.